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APPLICATION NOTE

Topographic Confocal Raman
Imaging of ArchaeologicalSamples

Topographic Confocal Raman Imaging of Archaeological Samples
Introduction
In archeological research, various specimens such as paintings, mummies,
antique objects or fabrics are regularlyinvestigated. Those samples are often
unique and require specialized treatment when analyzed. This applicationnote introduces topographic confocal
Raman Imaging as a flexible, noninvasive and non-destructive tool for thechemical and molecular characterization of archaeological specimens.
Working Principles
Confocal Raman Imaging
The Raman effect is based on lightinteracting with the chemical bondsof a sample. Due to vibrations in thechemical bonds this interaction causes
a specific energy shift in the back scattered light which appear in a unique
Raman spectrum through which themolecular components of a sample
can be detected. The confocal Raman
Imaging technique combines Raman
spectroscopy with a confocal microscope. Thus the spatial distribution of
the chemical components within thesample are imaged. High-resolution
confocal Raman microscopes acquire
the information of a complete Raman
spectrum at every image pixel andachieve a lateral resolution at thediffraction limit (circa λ/2 of the excitation wavelength). A confocal microscope setup is furthermore characterized by an excellent depth resolution
and features the generation of 3D
Raman images and depth profiles.
Confocal Raman microscopy can becoupled with correlative microscopy techniques such as fluorescencemicroscopy, electron microscopy andatomic force microscopy.

The Raman Principle

Topographic Confocal Raman Imaging
Raman topographic imaging is enabled with the TrueSurface option. 3Dchemical characterization on rough,inclined or irregularly-shaped samples
can be carried out precisely along asurface or at a set distance below
without requiring sample preparation.
Confocal Raman microscopes strongly
reject out of focus light. The TrueSurface module allows the surfaceof even large or coarsely textured
samples to remain in constant focus,
delivering the full benefits of the technique in terms of nanometer depth
resolution and stray light suppression.
As the sensor actively monitors andmaintains a set distance between
the objective and sample surface, itsclosed-loop operation can eliminate
any thermally-induced variations
during measurements with long integration times.

The application note was compiled in collaboration with Admir Masic, Department
of Civil and Environmental Engineering,MIT, Cambridge, MA, USA

The TrueSurface Principle

Raman spectroscopy has long been
employed as a spectroscopic tool formaterials characterization in the fieldof cultural heritage. Its non-invasiveand non-destructive nature make it anattractive candidate for both material
constituents and damage assessmentand monitoring. However, specificrequirements associated with objectsof interest such as the impossibility ofsampling as well as frequent autofluorescence associated with ageing
related degradation products, madethe extraction of useful Raman information extremely difficult.
In this application note it is shownhow to overcome some of the mentioned challenges by applying the mostadvanced Raman imaging techniques.
The sampling issue was addressed byapplying a large-area imaging approach in correlation with the state-ofthe-art profilometric measurement.
This patent-pending method wasrecently developed by WITec. In such a
way large objects (in terms of dimensions) can be automatically scannedand surface chemical images acquired.
The fluorescence problem is also an
important one and cannot be easily
solved. Recent advances in surface-en-hanced Raman spectroscopy (SERS)
enable the extraordinary enhancement of the Raman signal that canbe seen above the autofluorescence.
However, the limitation of the enhancement to only some types of the chemical compounds makes SERS specificto a limited number of applications.
Here we suggest another approachto address the fluorescence issue: It
combines the collection of the non-resonant Raman signal with a diffraction-limited spatial resolution. Thisapproach allows the spatial separationof fluorescence affected degradation
products and fluorescence-free intact
particles.

section of the sample was additionallyprepared and examined in order togenerate vertical depth-profiles of thesample layers in the z-direction.

from which the TrueSurface confocal
Raman imaging was consecutively
recorded is marked with a black rectangle.

Materials and Methods
All results were acquired with a WITecalpha500 R microscope for large-area
confocal Raman imaging. Furthermore
an Ultra-High-Throughput Spectrometer UHTS300 with a 600g/mm grating(BLZ 750nm) in combination with a785 nm excitation laser, a deep depletion CCD camera, and a 100x NA 0.9 air
objective was used. The TrueSurface®
topographic confocal Raman imaging
extension was directly integrated withthe objective turret, which allows theacquisition of topographic and Raman
data without any sample repositioning.
A piece of an ancient wall painting
served as the specimen for all investigations. The fragment, part of a private collection, displays several layersof original polychrome paintings inrelatively precarious state of preservation. The sample is shown in Fig. 1 withthe areas of interest that have beencharacterized indicated by the arrows.
For the investigation of the area ofinterest 1 the sample was not prepared prior to the measurements. Dueto the absorption of the sample thelaser light is unable to penetrate deep
through the layers. Therefore a smallFig. 2: White light stitching image of thearea of interest 1 of the sample.

Fig. 1: Photograph of the sample with theareas of interest labelled.

Results
Investigation of the Area of Interest 1:
It is clear from Fig. 1 that the area ofinterest 1 shows a strong topography
and can therefore not be measured byconventional confocal Raman microscopy. Therefore TrueSurface® confocal
Raman imaging was used to gather
the information from this area. In thismode the topography is first recorded
using an optical profilometer. By simply rotating the objective turret thesystem is changed to Raman modeand the recorded surface can then beused to ensure that the sample always
stays in focus.
Prior to the measurement a whitelight stitching image was recorded tolocate the area of interest. The whitelight image is shown in Fig. 2, the areaFig. 3 shows the sample’s topography
recorded over an area of 5x10mm2with the TrueSurface® profilometricsensor. Please note that a change ofover 500 µm in the focus position
was observed. This variation in height
would normally prevent measurements in standard confocal Raman
imaging mode due to the surface
being frequently out of focus.
Following the recording of the surface
a large area confocal Raman image
was recorded using the topographic
information to trace the z-position.
The scanned area in Raman imaging mode was again 5x10mm2 with
150x300 Raman spectra recorded. The
integration time per spectrum was 1s.
The most prominent red painting layer
in the analyzed area could easily beidentified as cinnabar (HgS). Cinnabar
is a precious red pigment characteristic of the Roman period. Minium,
a ground version of cinnabar, wasconsidered the Romans’ most valuable pigment and it was used in many

aFig. 4: a) White light image of the area ofinterest 1 with the locations at which cinnabar pigment could be found overlaid inred. b) Photograph of the area of interest 1.
Fig. 3: Topography of the sample as recorded using the TrueSurface® extension of
the alpha300 microscope series.

ambitious works, including some
Pompeiian masterpieces, and demonstrated great wealth. In fact, the priceof Spanish minium used in Pompeii
was capped by law at 150 sesterces
per kilogram to stop it rising out of all
proportion (Pigment Compendium: A
Dictionary and Optical Microscopy ofHistorical Pigments By Nicholas Eastaugh, Valentine Walsh, Tracey Chaplin,
Ruth Siddal 2008, Elsevier).The location at which cinnabar was detected by
confocal Raman imaging is overlaid in
red onto the white light image in Fig.
4a. By comparing the location of thecinnabar with the red painted areas
shown in Fig. 4b, it can be clearly seenthat the distribution of cinnabar isclosely correlated. Furthermore an appearance of cinnabar can be detected
at the edges of the layers indicatingthat a cinnabar-based preparation hadbeen applied which evinces the importance and richness of the analyzed
fragment.
In addition to cinnabar, other components of the investigated area can beb

identified by their unique Raman spectra. Fig. 5 shows the primary spectral
components of the Raman analysis.
Thus calcite, carbon black, and an
unknown phase can be additionallydetected.
Analyzing the entire data set allowed
the generation of a color-coded image
which could then be overlaid onto thetopography as shown in Fig. 6. The
colors of the areas correspond to thecolors of the spectra shown in Fig 5.

Fig. 5: Spectral components Raman analysis. The spectra could be identified as: Red= Cinnabar; Green = White Chalk (CaCO3,Calcite); Blue = Carbon Black; Turquoise =unknown phase.

Fig 6: Color-coded Raman Image
overlaid onto the topography ofthe area of interest 1. The colorsof the image correspond to thecolors in Fig. 5.

Investigation of the Area of Interest 2:
Next the area of interest 2 was investigated. Here a distinctly different redcolor can be seen compared to thebright red color investigated in areaof interest 1. This observation could beconfirmed by the spectral analysis. The
Raman spectra shown in Fig. 7 identifies red ocher as the main chemicalcomponent of the dark red color ofarea of interest 2. For comparison thespectrum of cinnabar is also shown inFig. 7.

Fig. 8: Color-coded Raman
image overlaid on thewhite light image of thearea. Color coding: Ocher
crystals - Red; Calcium carbonate binder mixed withocher - Blue; High level offluorescence - Green.

The color-coded Raman image wasoverlaid onto the white light image asshown in Fig. 8. The colors correspondto the colors of the spectra shown inFig. 9 with ocher crystals shown in redand calcium carbonate binder in blueand green, indicating a high level offluorescence.
Fig. 7: Spectra of the bright red color (identified as minium) and of the dark red area
(identified as red ocher)

For the generation of a confocal
Raman image of area of interest 2
an area of 70x70µm2 was measured
with an integration time of 133ms
per spectrum. The resulting Raman
image consists of 70x70 pixels with
the information of a complete Raman spectrum at every image pixel.

Fig. 9: Spectra extracted from the confocal
Raman image scan on area of interest 2.
The colors correspond to the colors shownin Fig. 8.

Cross Section Measurements
As outlined earlier, the penetration
depth of the laser light into the material is very limited. For this reason
Fig. 10: Color-coded
confocal Raman
image overlaid on
a white light imageof a cross sectionof the sample. Thecolors correspondto the colors of thespectra in Fig. 11.
The frame indicatesthe area at whicha consecutivelysecond scan wasperformed.

cross sections were prepared. The
cross sections allow the visualizationof the succession of individual layers
in z-direction (vertically). Therefore asmall sampling is necessary and thefragment taken is embedded in a resin
then polished perpendicular to theplane of the painting surface. In thisway a suitable specimen cross section is obtained for observation of theindividual layers by confocal Raman
microscopy.

Fig. 11: Spectra extracted from the confocal
Raman image scan shown in Fig. 10. Greenand blue: calcite in two different configurations/orientations; Red: Minium; Yellow:Amorphous carbon.

Fig. 10 shows the overlay of the combined Raman image onto the whitelight image. The Raman image wasrecorded with 175x175µm2 (175x175pixels). The integration time was
133ms per spectrum and the light wascollected through a pinhole with a diameter of 100µm. The gray frame in Fig.
10 shows the zoomed area that wasrecorded in a second scan. The colorsof the overlay image correspond tothe colors of the Raman spectra in Fig.
11. The spectra in Fig. 11 show calcite
characteristic profiles in two different
configurations/orientations (greenand blue spectra). The minium is again
identified as the red pigment used(red spectrum) as well as a very weaksignal of amorphous carbon originating from a thin layer at the surface(yellow color).

With a highresolution
scan (markedwith a grayframe in Fig.
10) the area
at which theminium color
particles canbe prominently seen
was imaged
again. Fig. 12shows thecombinedcolor-coded
Raman imageand Fig. 13 thecorresponding spectra.
In this areaa furthercomponentof unknown
origin (bluecolor) could be extracted from thespectra using spectral de-mixing.
From the image it can be seen thatthe color particles (in Red) cluster intolarger and smaller agglomerates andthe smallest detectable in the image
are on the order of half a micrometer (marked with an arrow in Fig. 12)
which corresponds to the resolution
limit for a 785nm laser used in conjunction with a 100X NA 0.9 objective(0.51* λ/NA ≈ 445nm [FWHM]).
Conclusion

Fig. 13: Raman Spectra extracted from thezoomed scan of the cross section. Red: Minium; Blue: Unknown origin; Green: Calcite

The recent instrumental developments in Raman microscopy enableremarkable new applications inarchaeological sciences. The possibility
given by TrueSurface® of non-invasively and non-destructively studying
large areas of ancient objects withirregular surfaces is a breakthrough inthe field of cultural heritage research. It further allows investigation

Fig. 12: Combined Raman image of thezoomed scan of the cross section. The
colors correspond to the colors of the spectra shown in Fig. 13. The arrows mark thesmall agglomerates on the order of half amicrometer.

of extremely precious samples in situ
without the need for any sampling orsample preparation. When samplingis possible, the sub-micron resolution
large area imaging of a cross section
further improves the quality of theanalytical output and allows detailed
characterization of primary material
constituents on the micron scale. In
conclusion, confocal Raman imaging
is a valuable tool for the identificationof minute quantities of a material
that can provide the archaeologist,
conservator and other researcherswith invaluable information regarding
provenance, manufacturing technology, state of preservation, and in someinstances, authenticity.